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ABSTRACT: Characterization of the molecular response under caries lesions requires a robust and reliable transcript isolation system, and analysis of data indicated that collection of extracted teeth in either liquid nitrogen/RNA-stabilizing solution facilitated this. Subsequent transcriptional analysis indicated higher general activity in carious pulps, while characterization of inflammatory mediators, including cytokines and S100 proteins, highlighted increasing expression levels associated with both microbial front progression and elevated cellular immune response. Analysis of the pleiotropic hormone adrenomedullin (ADM) indicated that transcript and protein levels are increased in pulpal tissue during caries, and that protein levels sequestered in dentin due to primary dentinogenesis are comparable with those of TGF-β1. Expression analysis of a leucine-rich-repeat-containing protein (LRRC15/Lib) indicated that this highly conserved molecule was up-regulated during caries, is transcriptionally regulated by pro-inflammatory stimuli, and is relatively abundant in mineralized tissues.
Advances in dental research 07/2011; 23(3):290-5.
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ABSTRACT: The odontoblast is the secretory cell responsible for primary, secondary and tertiary reactionary dentinogenesis. We provide evidence that the changes in secretory activity of odontoblasts reflect differential transcriptional control and that common regulatory processes may exist between dentine and bone.
Based on the hypothesis that differential dentine secretion (primary and secondary dentinogenesis) is associated with changes in the transcriptional control within the cell, we have investigated the transcriptome of odontoblasts at young and mature stages and subsequently used this information to identify key regulatory intracellular pathways involved in this process.
We used microarray analysis to compare the transcriptome of early stage (primary dentinogenesis) and late stage (secondary dentinogenesis) odontoblasts from 30 month old bovine teeth. Secondarily, we used post-array sqRT-PCR to confirm the differential expression of 23 genes in both populations of odontoblasts. Finally, immunohistochemistry was performed on bovine and murine tissues with antibodies to DMP1 and anti-phospho p38 proteins.
DMP-1 and osteocalcin gene expression were up-regulated in the mature odontoblasts, whereas collagen I, DSPP, TGF-beta1 and TGF-beta1R gene expression were down-regulated. Microarray analysis highlighted 574 differentially regulated genes (fold change>2 - p<0.05). This study supports further existing similarities between pulp cells and bone cells. Using post-array Sq-RT-PCR we characterized transcript levels of genes involved in the p38 MAP kinase pathway (PTPRR, NTRKK2, MAPK13, MAP2K6, MKK3). Differential p38 gene activation was confirmed by immunohistochemistry for p38 protein in murine teeth. Finally, immunohistochemistry for DMP1 indicated that odontoblasts involved in primary and secondary dentinogenesis may coexist in the same tooth.
As established in bone cells, the transcriptome of the odontoblast was shown here to evolve with their stage and functional maturity. Identification of the involved signalling pathways, as highlighted for p38, will enable the deciphering of physiology and pathology of mineralised tissue formation.
Bone 07/2009; 45(4):693-703. · 4.02 Impact Factor
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ABSTRACT: Aim To determine whether odontoblasts demonstrate differential gene expression during primary and secondary dentinogenesis. Methodology Coronal pulps were removed from immature developing and mature erupted bovine teeth extracted from 30-month-old animals. RNA from odontoblasts was selectively extracted [McLachlan et al (2003) Archives of Oral Biology48, 373-83]. Microarray analysis (Affymetrix) was used to compare the transcriptomes between the two odontoblast populations. One hundred nanograms of total RNA were initially double amplified to generate cDNA. Twenty-five micrograms of the cRNA generated by in vitro transcription reaction was fragmented using 5x Fragmentation buffer and RNase-free water at 94 degrees C for 35 min to generate 35-200 base fragments. Fifteen micrograms of fragmented cRNA was reconstitued in a hybridization cocktail, which was applied to each array and hybridized for 16 h at 45 degrees C. The two samples were analysed in duplicate using four GeneChip Bovine Genome arrays (900561). Bioinformatic analysis was performed using three software programmes (D-chip, Onto-Express and Pathway express). In parallel, the expression of 20 genes were compared between two populations of odontoblasts by RT-PCR [Early stage (ES) and late stage (LS)] (ADM, AMEL, BMP4, Clock Genes, CollagenIII, CollagenI, DMPI, DSPP, MEPE, Msx1, Msx2, OSTEOCALCIN, PGAP I, TGFbeta1, TGFbeta1R, NESTIN, Alkaline Phosphatase, SHH, NaNog, LPR15). Results Of the 24 000 genes analysed by microarray, 1338 were differently expressed in both populations by at least twofold (Paired analysis, P < 0.05). A total of 764 genes were up-regulated and 574 down-regulated in ES compared with LS odontoblasts. Amongst these genes, several known markers of odontoblasts (including Coll I, DMP1, Amelogenin, Osteoclacin) were found to be differentially expressed, as weel as several other genes not previously studied in odontoblasts. Several of these are components of intracellular signalling, including pathways involved in MAPKinase, apoptosis, TGF-Beta, or FGF transduction. Ontological analysis revealed that 28.33% of the bovine genes identified by microarray analysis were assignable to biological processes, 26.79% as cellular components and 32.26% in molecular processes. RT-PCR analysis confirmed the differential expression of some of the cited genes. For example, Amelogenin was only detected in ES, whereas osteocalcin was detected only in LS odontoblasts. Collagen-I expression was not, however, differentially expressed, although Adrenomedullin and DMP1 were, up- and down-regulated in LS, respectively. These results agreed with the microarray findings. Conclusion The differential changes in gene expression in primary and secondary dentinogenesis indicate modifications in transcriptional control of the cells and highlight the need to identify the nature of these control mechanisms both to characterize cell phenotype and to better understand how cell secretory behaviour can be controlled during tertiary dentinogenesis.
International Endodontic Journal 10/2008; 41(9):815-6. · 2.18 Impact Factor
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ABSTRACT: To assess the feasibility of using the mouse as an in vivo model for studying pulpal healing in response to restorative procedures.
Direct pulp capping on maxillary first molar teeth with mineral trioxide aggregate (MTA), overlaid with light-cured composite resin, was performed on nineteen 3-month-old mice. For control teeth, the composite resin was placed in direct contact with the pulp. Animals were killed at 3 days, 1 week, 2 weeks, 5 weeks and 11 weeks postoperatively. Extracted dental tissues were subsequently analysed by haematoxylin and eosin staining, immunohistochemistry for dentine sialophosphoprotein (DSPP) expression, scanning electronic microscopy and X-ray analysis to determine both pulpal response and dentine bridge formation.
Of the 19 mice initially used, 16 were subsequently studied. Histological analyses of pulps directly exposed to MTA for up to 2 weeks demonstrated a distinct structural change in the extracellular matrix. By weeks 5 and 11, a dentine bridge was present in all MTA-treated specimens in which DSPP immunoreactivity was clearly apparent. Scanning electronic microscopy and X-ray analysis enabled confirmation of calcification of the dentine bridge, and demonstrated that it had a globular surface morphology as opposed to the tubular appearance associated with orthodentine.
This is the first description of the utilization of a murine model for study of in vivo pulpal repair. This approach provides a novel opportunity to enable the use of genetically modified animals to explore cellular and molecular processes during reparative events.
International Endodontic Journal 10/2008; 41(9):781-90. · 2.18 Impact Factor
